Methods

Purification of Microtubular Proteins-The initial stage of puri-fication was that of Basford (9). To ensure the structural integrity of mitochondria and synaptosomes, the extraction of cytosolic proteins was performed with a buffer having a tonicity of about 600 mosm. Bovine brains were stripped of meninges and 150 g of gray matter removed with scissors.(It is apparently unnecessary to free the gray matter completely of the white matter.) To this tissue was added 1 ml of extraction medium (0.52 M sucrose, 1 mM EGTA, 1 mM ATP, pH 7.0 with KOH)/g of wet tissue. The tissue was homogenized in a 50-ml glass-Teflon homogenizer with at least five passes of the pestle, and the homogenate was centrifuged at 75,000 X g for 60 min at 4 C. The crude extract (CE fraction) was diluted 10% with concentrated buffer (10 X MEM buffer, see below) containing 1 mM GTP to give a final concentration of 0.1 M 2-(N-morpholino) ethanesulfonic acid, 1 rnM EGTA, 1 mM MgC12, pH 6.8 (MEM buffer) with 0.1 mM GTP. Microtubules were further purified using a modification of the Shelanski method (6). First, glycerol was added to 3.5 M, and polymerization was initiated by warming to 37 C. After a 45. min incubation period, microtubules were collected by centrifugation at 75,000 x g for 90 min at 37 C. The pellets were then resuspended in MEM buffer containing 1 mM GTP at 4” C, homogenized gently with one pass in a glass-Teflon homogenizer(HIP fraction), depolymerized on ice for 30 min, and centrifuged for 60 min at 75, ooO x g and 4 C. The supernatant (CIS fraction) was brought to 3.5 M glycerol and was then polymerized for 45 min by raising the temperature to 37 C. Microtubules were pelleted by centrifugation(60 min, 75,000 X g, giving the HIP fraction), and this protein was subjected to another cycle of polymerization/depolymerization as described above (giving the CB and H, P fractions). Protein was stored at-80 C in pellet form after rapid freezing in liquid nitrogen. At each step of the purification procedure, aliquots were withdrawn for protein determination and electrophoresis(see below).